KR960005086B1 - Method of cultivation of microorganism ice-nucleation - Google Patents

Abstract

When pseudomonas syringae(KCTC 1832) is cultivated in the fermentor including industrial microorganisms, yeast extract, soy pepton, and corn-steep liquor, the following processes can increase the productivity of the microorganism. (a) the process of limiting dissolved oxygen to 10-50 % with oxygen membrane; (b) the process of providing medium to maintain above 1% of glucose concentration; (c) the process of lowering culture temp. to 20-24 deg.C with dissolved oxygen maintained below 1ppm.

Description

Cultivation method of microorganisms having ice nucleus activity

1 is a graph showing that in the present invention, lactic acid contained in the initial medium is consumed by Pseudomonas syringe KCTC 1832 even in the presence of glucose,

2 is a graph showing the change in ice nuclei activity of each class when the dilution rate (same as growth rate) is 0.04hr ⁻¹ and the temperature is decreased from 28 ° C. to 22 ° C. in the carbon source limited continuous culture in the present invention.

The present invention relates to a method for culturing by high-productivity fermentation of Pseudomonas syringae, a microorganism having ice-nucleus activity, and more specifically, to the high-cost production of Pseudomonas syringe, a microorganism containing ice-nucleated protein, at high concentration in a fermentation tank. The present invention relates to an improved culture method for culturing by batch culture and continuous culture including culture.

Here, productivity refers to the concentration in the fermentation tank (g. / L) of the microbial cells having ice nucleus activity, the amount of cells to be produced per unit time or volume of the fermentation tank (g / Lhr), and the number of nuclei of ice nucleus activity produced per unit cell ( active nuclei numbers / g cell).

Microorganisms with ice nucleus activity have been known since the early 1970s, mainly around Pseudomonas, Erwinia and Xanthomonas. Ice nucleation activity is due to ice nucleation protein (INP) produced by microorganisms. It is known that the activity occurs in the unique structure of ice nucleus activating protein, which is applied to ice making, snow removal and freezing air conditioning industry, food industry, development of strains to prevent cold damage, or development of effective diagnostic reagents through identification of its mechanism of action. Research is underway (Wolber and Warren, "Bacterial ice-nucleation protein" (Trends in Biochemical Science 14: 179-182, 1989)).

In order to industrially use ice nucleating microorganisms having such an application range for any purpose, a technology capable of mass production is required. The mass production fermentation technology can be divided into two perspectives. The first is a fermentation technology for mass production of microbial cells having ice nucleus activity, and the second is to adjust the fermentation environment to maximize the ice nuclei activity per cell.

Related technologies include culture of Erwinia uredova and Pseudomonas viridiflava (Japanese Patent Publication No. 63-230473), Pseudomonas syringae ACTC 53543 The media conditions (US Pat. No. 944120), pH control (US Pat. No. 910600) and two-stage temperature control method (US Pat. No. 21949) are known.

However, these techniques have a problem that the productivity and cell concentration of the fermenter is low.

Therefore, in the present invention, in order to overcome the problems of the conventional techniques as described above and mass production and to maximize the ice nucleus activity per cell, an optimization technique through high concentration culture and continuous culture was introduced.

In other words, increasing the final cell concentration in fed-batch cultures and repeated fed-batch cultures not only improves fermenter use efficiency, but also does not require a concentration step that requires the use of a continuous centrifuge or membrane separator. Allow to lyophilize.

In addition, the production of ice nucleus active microorganisms using high concentration continuous culture not only improves productivity but also has homogeneous ice nucleus activity. When the dissolved oxygen is insufficient in continuous culture and fed-batch culture, oxygen enrichment membrane [Hungyang Co., Ltd.] , Seoul] solved the problem by supplying air with increased oxygen partial pressure. In addition, to maximize ice nuclei activity per unit cell, optimal fermentation environment was established through physiological studies of microorganisms in abnormal state in continuous culture.

Therefore, the present invention is to produce a microbial cell mass through the batch culture and continuous culture method of the Pseudomonas Syringe microbial having ice nucleus activity, and ice nucleus activity to control the fermentation environment to maximize the ice nuclei activity per cell Its purpose is to provide a method for culturing microorganisms.

Hereinafter, the present invention will be described in detail.

The present invention is a method for culturing ice nucleus activated microorganisms, using the ice nucleus activated microorganism Pseudomonas syringe KCTC 1832, the milk value culture to produce ice nucleus active protein-containing microorganisms in a fermenter at a high concentration of 35 ~ 100g / l (dry cell concentration) It characterized by culturing by batch culture or continuous culture, including. However, the microorganisms used should not be limited to Pseudomonas syringe KCTC 1832, but should be considered as a general method of high concentration culture of Pseudomonas syringe.

In the present invention, in culturing Pseudomonas syringae KCTC 1832, a microorganism having ice nucleus activity, the microorganism is cultured in an industrial microorganism-containing fermenter containing corn steep liquor, yeast extract and soy peptone (a). Process of limiting dissolved oxygen to keep dissolved oxygen 10 ~ 50% by using oxygen enrichment membrane during the process

(b) supplying the culture medium so that the concentration of glucose in the fermenter is not less than 1%; and

(c) characterized in that the culture in one or more processes selected from the step of lowering the culture temperature to 20 ~ 24 ℃ in a state in which dissolved oxygen is limited to less than 1ppm.

In the present invention, in order to produce a high concentration of microbial cells in the oil value cultivation and continuous culture in order to (1) supply a balanced nutrient source, (2) solve the problem of dissolved oxygen deficiency, (3) There are four main problems: prevention of temperature rise due to (4) prevention of accumulation of ancillary by-products (introduced in fermentation medium and produced during growth) which may inhibit the growth of microorganisms.

Hereinafter, the present invention will be described in more detail through a method for solving the above four problems.

First, the medium design for the supply of balanced nutrients is the most important of the fermentation optimization. The basic design in the present invention is described in SJ Pirt's book "Principles of Microbes and Cell Cultivation" 1975, Blackwel Ltd., 134. It is mainly referred to as a method based on the chemical analysis of the microbial cell composition, the medium is designed to suit the cell composition. Therefore, in the newly isolated strain in nature there is no information on the growth conditions or media components can be useful in such a medium design.

The strain used in the present invention corresponds to this, and the strain used in the present invention is Pseudomonas syringe AL (P. syringae AL; Kim Jung-han et al., 1989, Korean Journal of Biochemistry 22: 73-77), isolated from domestic soil. 1832, which was deposited in the Genetic Bank of Korea Institute of Science and Technology.

For the microorganism of the present invention, yeast extract, peptone, soyton, protein hydrolyzate, corn-steep liquor, and the like were added as an organic compound to the basic medium design based on chemical composition.

The composition example of each component was specified in the Example.

Second, the solution of dissolved oxygen deficiency problem is one of the most basic problems to be solved for high concentration culture of microorganisms. The lack of oxygen necessary for the metabolism of microorganisms causes problems such as partial inhibition of metabolic processes and the production of ancillary products.

In order to improve the oxygen transfer capacity of the fermenter, when the aeration rate and stirring speed are maximized and the dissolved oxygen is insufficient at less than 1ppm, the oxygen enrichment membrane device that can increase the partial pressure of oxygen in the air is used. The oxygen partial pressure was increased to 45-50% and used for ventilation.

Oxygen enrichment membrane device is used to increase the partial pressure of oxygen in the air by membrane separation technology.

In addition, when the oxygen partial pressure is increased with air or oxygen enrichment membrane, and the dissolved oxygen can no longer be maintained, a method for reducing the oxygen demand of the microorganism by lowering the medium supply rate is also used. If the dissolved oxygen could not be maintained above 1 ppm by any method, the medium feed rate was adjusted so that the growth of the oxygen-restricted state continued. At this time, all oxygen supplied from the aeration is used for metabolism by the microorganisms so that the dissolved oxygen concentration becomes zero, and the growth of the microorganism continues as much as the supplied oxygen.

Third, the metabolic heat produced by the growth of microorganisms is not a problem at low concentrations as in the case of normal fermenters. When culturing at a high concentration as in the present invention, specially cooled water or a temperature controller having a cooling circulation system should be used. In the present invention, the latter was used.

Fourth, the accumulation of by-products that can inhibit the growth of microorganisms can be divided into the accumulation of components not used by the microorganisms in the medium that is continuously introduced and the accumulation of ancillary products generated during the growth of the microorganisms. The lactic acid contained in the corn steep liquor used in the present invention corresponds to the former case. When Pseudomonas syringe KCTC 1832 used in the present invention was cultured in a medium containing glucose and corn steep liquor (see Examples 1 and 2), lactic acid was used by this strain even in the presence of glucose (FIG. 1). That means that even if the strain is capable of producing lactic acid, it can be used again under environmental control.

Although many fermentation experiments have been carried out for the present invention, it has been confirmed by the glucose / lactic acid analyzer that no lactic acid accumulates. In addition, as many strains of Monas monas strains are known to form polymer polysaccharides, polymer polysaccharides have been observed to be produced when the physiological state of the oxygen-restricted condition is continued even in the present invention. However, if too many polymer polysaccharides are produced, the rare elements in the media may be aggregated, which may affect the growth of microorganisms, but such polymers are not produced under the optimal conditions established by the present invention.

In the present invention, the problem of maximizing the ice nucleus activity produced per produced microbial cells is another problem of producing microbial cells themselves, and the method used to maximize the ice nuclei activity per unit cell is abnormal in continuous culture. Microbial physiology optimization experiment through state. This method was used because it is possible to obtain accurate results on the effects of fermentation environment variables under conditions known to limit substrate and growth rate (usually difficult to maintain a constant environment in batch culture).

2 shows the change in ice-nuclei number per unit cell when the dilution rate is kept constant at 0.04hr ^-1 and the temperature is changed from 28 ℃ to 22 ℃ in a carbon source-limited continuous culture tank. It is not measured. As can be seen in FIG. 2, the effect could be measured within a few hours, and in addition to the temperature change effect shown in FIG. 2, similar experimental results were optimized for limiting substrate, dissolved oxygen concentration, temperature medium type, and the like. Applied in the case of.

Temperature is one of the most important factors in maximizing ice nucleation activity. Ice nucleation activity is improved below 25 ° C, and ice nucleation activity is not much improved even lower than 20 ° C.

Also noteworthy in FIG. 2 are activities classified by classes A, B, and C (representing activity at 4.4 ° C or higher, -4.8 to -5.7, or -7.6 or higher, respectively) according to ice core activation temperature. It is true that the change of can be measured separately. Ice nucleus water used the method of Vali, which was measured by continuously diluting the solution adjusted to absorbance 1.0 by 10 times (J. Atmos. Sci., Vol. 28, 402-409, "Quantitative evaluation of experimental results on the heterogeneous freezing of supercooled liquids ").

In order to increase the ice core active water per unit cell, it was found that it is advantageous for the microorganism to grow at a low rate (less than 1/3 of the maximum growth rate). In the present invention, the growth rate is mainly limited by the limited supply of nutrients required by the microorganism. Lowered.

Here, the limited supply is to lower the substrate advanced speed than the maximum substrate consumption rate of microorganisms, the ratio can be adjusted to the desired speed by adjusting the speed of the pump for supplying the culture medium.

In the present invention, the experiment was conducted mainly in the case of obtaining the maximum number of ice cores, the maximum number of ice cores having a similar number of ice cores compared to the number of microorganisms in the liquid at the absorbance 1.0 (4.5 × 10 cells / mL, ± 20%) Can be said to have reached the maximum number of ice cores. Because even if there is more than one ice nucleus per microorganism, one microorganism cannot be divided into two by simple dilution in the experimental technique. Therefore, if all ice nuclei exist in each microorganism, that is, the maximum number of ice nuclei in a liquid. It is.

Therefore, the microorganism Pseudomonas syringe having ice nucleus activity can be produced at high concentration in a fermenter using the method of the present invention, which can be applied to the development of strains for the prevention of cold damage or to the development of effective diagnostic reagents, artificial ice making and snowmaking, refrigeration industry and processing industry It can be widely used in various fields such as.

Hereinafter, the present invention will be described in detail with reference to Examples, but the present invention is not limited by Examples.

[Reference Example]

The plate was stored in a 4 ° C. refrigerator in an LB medium and inoculated into a 500 ml baffle-lined triangular flask containing 50 ml of Elby or TPB medium at 150 rpm in a shaker at 30 ° C. for 12-15 hours. Those cultured were used as inoculation bacteria in fermenters. Inoculation was 3-10% and fermenter was adjusted to 5-L size and 2.5-3.0L of actual operating volume.

After starting the fermentation, glucose and lactic acid were measured using a glucose-lactic acid analyzer (YSI 2300, U. S. A), and cell concentrations were measured after 105 ° C. drying or lyophilization, in addition to the method of measuring the absorbance in a short time. The pH of the culture was adjusted to 6.0-6.5 using 30% ammonia water. Aeration rate was adjusted from 0.5v / v / m to 2.0v / v / m according to the change of dissolved oxygen and the stirring speed was adjusted to 500-1500rpm to maintain 15% or more dissolved oxygen except in special cases.

If the oxygen demand is so high that dissolved oxygen cannot be maintained even at the highest aeration rate and agitation rate (more than 25-35 g / L of cell concentration), the feed rate of the limiting medium is controlled to lower the oxygen demand or fermentation with limited oxygen. Was continued, and in some cases, an oxygen enrichment membrane was used to supply concentrated air to contain 45% oxygen. When the cells were produced at a high concentration of 50 g / L or more, they were lyophilized immediately without a concentration step or washed by a continuous centrifuge or micro filtration to remove deposits, and then lyophilized.

Example 1

The initial medium of this culture tank was 4% corn steep liquor, 1% yeast extract, 0.5% soypeptone, 1% calcium phosphate, 1% hydrous glucose and the initial pH was adjusted to 6.7 using 2N NaOH. 30 minutes sterilization (glucose separately sterilized) was inoculated under the above conditions to start fermentation. The temperature was maintained at 25 ° C and cultured. After 20 hours, the solution prepared with 10% glucose 40% corn steep liquor, 1% soypeptone, and 1% yeast extract was supplied to the fermenter so that the glucose concentration was not lower than 1%. When the culture was continued for 40 hours, a dry cell concentration of 62 g / L was obtained.

Example 2

The initial medium of this culture was 5% corn steep liquor, 1% yeast extract, 0.5% soy peptone, 1% calcium phosphate, 1% hydrolyzed glucose, and 40% glucose and corn steep liquor 10 hours after incubation. The dry cell concentration of 81 g / L was obtained at 50 hours when the liquid continuously prepared at 10% was supplied.

From the cell concentration of 21 g / L (15 hours of incubation time), the dissolved oxygen concentration dropped to 5% or less and continued until the growth of the oxygen-limited state. Maintaining the culture temperature at 26 ℃ until the limit growth by dissolved oxygen, and when going to the limit growth stage, the incubation temperature was lowered to 21 ℃ to continue the cultivation to complete limit growth.

Example 3

The initial medium of this culture tank was 4% corn steep liquor, 1% yeast extract, 0.5% soypeptone, 1% calcium phosphate, 5.5% hydrochloride, and the temperature was started at 22 ° C.

The additional feed medium was 96 g / l dry cell concentration after 46 hours of cultivation using a solution composed of 50% glucose, 10-% corn steep liquor, 1% soypeptone, 1% yeast extract, and 5% ammonium sulfate. Could get In this case, the aeration was continued through 45% oxygen-containing air through the oxygen enrichment membrane (heungyang) after 18 hours of culture.

Example 4

The initial medium of this culture tank was incubated at 25 ° C. as a semi-synthetic medium without using a complete complex medium as in Examples 1 to 3. The initial medium was 77 (g / L) glucose, MgSO ₄ 7H ₂ O 2.3, NH ₄ Cl 10, NaNO ₃ 3, KH ₂ PO ₄ 11.3, CaCl ₂ 2H ₂ O0.1, FeSO ₄ · 7H ₂ O Cultivation was started by the composition of 0.07, MnSO ₄ · 5H ₂ O 0.02, ZnSO ₄ · 7H ₂ O 0.02, yeast extract 5.0, Soyton 2.5, Tri-Na citrate 2. From 36 hours of culture, 60 hours of feeding was continued with 50% glucose, MgSO ₄ · 7H ₂ O 2.3%, NH ₄ Cl 1%, KH ₂ PO ₄ 1.13%, and yeast extract 3%. As a result, a dry cell concentration of 88 g / L was obtained. Incubation was continued by lowering the incubation temperature to 22 ° C with additional media.

The examples thus far exemplify the productivity improvement through fed-batch culture and the following examples relate to high concentration continuous culture. The advantage of the production by continuous culture is that in addition to being able to produce microorganisms having a constant activity than fed-batch culture, the amount of cells that can be produced in a unit fermenter, that is, productivity is increased.

EXAMPLES 5-12

Continuous culture was performed by determining the initial medium. Glucose 77 (g / L), MgSO ₄ · 7H ₂ O 2.3, NH ₄ Cl 10, KH ₂ PO ₄ 11, CACl ₂ · 2H ₂ O 0.1, FeSO ₄ · 7H ₂ O 0.07 The culture was started with MnSO ₄ · 5H ₂ O 0.02, ZnSO ₄ · 7H ₂ O 0.02, CuSO ₄ · 5H ₂ O 0.005, yeast extract 5.0-20.0, Soyton 2.5, Tri-Na citrate 2. The medium used for continuous culture was water-containing glucose 220 (g / L), MgSO ₄ · 7H ₂ O 2.3, NH ₄ Cl 20, NaNO 3 5, KH ₂ PO ₄ 11, CaCl ₂ · 2H ₂ O 0.1, FeSO ₄ 7H ₂ O 0.07, MnSO ₄ 5H ₂ O 0.02, ZnSO ₄ 7H ₂ O 0.02, CuSO ₄ 5H ₂ O 0.005, yeast extract 5.0, Soyton 2.5, Tri-Na citrate 2, and the like. The results of dry cell production for each dilution rate and temperature are shown in the following table.

Claims (1)

In culturing Pseudomonas syringae KCTC 1832, a microorganism having ice nucleus activity, the microorganism was cultivated in an industrial microorganism containing fermenter containing corn steep liquor, yeast extract and soy peptone (a) oxygen enrichment during the culturing process. Limiting dissolved oxygen to maintain 10-50% of dissolved oxygen using a membrane; (b) supplying a culture solution so that the concentration of glucose does not reach 1% or less in the fermenter; and (c) the dissolved oxygen is limited to 1 ppm or less. High concentration culture method of microorganisms having ice-nuclear activity, characterized in that the culture in one or more processes selected from the steps of lowering the culture temperature to 20 ~ 24 ℃.